Impact Of CO₂ Pneumoperitoneum Used In Laparoscopy On Endometrial Cells Proliferation In Vitro

Background:Endometriosis is a common gynecological problem. The pathogenesis of endometriosis remains unknown. The broken balance between proliferation and apoptosis of eutopic endometria plays a great role in the pathogenesis of endometriosis. Lack of adequate immune surveillance in the peritoneum is thought to be another important factor. Laparoscopy is the golden criterion for endometriosis diagnosis, which may be more effective than laparotomy in detecting and eliminating small lesions which are more easily visible during laparoscopy. Laparoscopic surgery becomes the first option to endometriosis patients.Compared with laparotomy, laparoscopy has resulted in a dramatic decrease in hospital stay, an increase in patient's comfort, and a more rapid return to normal daily activities. Since Dobronte reported the pneu-needle and trocar metastasis happened to a patient after laparoscopy in 1978, the researchers and the doctors have paid more and more attentions to the laparoscopic operations. Carbon dioxide (CO₂) is the most commonly used as insufflation gas to provide exposure in the abdominal cavity. It is one of the most hotly debated issues in laparoscopic surgery that the performances of oncological surgery use a CO₂ pneumoperitoneum as an alternative to the conventional approach. Lots of researches suggested that one of the important reasonsis using carbon dioxide pneumoperitoneum in laparoscopic tumor surgery, which changes the microenvironment of the peritoneal cavity. It could down-regulate the pH value, damage the structure of peritoneum, and especially be harmful to the macrophage in the peritoneal cavity that can clear the tumor cells in the cavity.EMs is a benign disease, but it has abnormal biological behaviors in invasion, proliferation and apoptosis. The lesions sometimes are too small to find out, and as usually some endometrial cells may survive at the implanted site after surgery, which may become a cyst or foci again. However the laparoscopy plays an important role in the diagnosis and treatment of EMs, researches about the impact of CO₂ pneumoperitoneum on ectopic and eutopic endometria are rarely reported. So our purpose is to investigate the impact of CO₂ pneumoperitoneum on the proliferation of the ectopic and eutopic endometrial cells based on the abnormality of eutopic endometria of EMs patients.Materials and Methods:Subjects undergoing elective surgery for ovary endometriosis were recruited for cyst wall (n=10), women undergoing hysterectomy surgery and pathology confirmed endometriomas were recruited for endometria (n=19), and the women undergoing hysterectomy surgery and pathology confirmed leiomyoma of uterus were recruited for endometria (n=12). Firstly, primary cell cultures were performed using immunohistochemistry to identify endometrial stromal cells and glandular epithelium cells. Secondly these cells were divided into two groups. One was maintained in a sterile environment in special drainage packs lasting for 90min. The environment was a model of CO₂ pneumoperitoneum used in the real laparoscopic surgery, which was full of 99.99% CO₂ and the pressure maintained at 15mmHg. The other group was put into the incubator the same as that the first group was put into. After CO₂ pneumoperitoneum, get the pH value of the media at once, and pH values were observed every 10min until it turned back to 7.0. Then we collected the cells from two groups at 24h, 48h after pneumoperitoneum. As the limitation of ectopic endometrial cells, we collected the cells only at the 48h after treatments. Fixing cellswith 70% alcohol was prepared for the flow cytometry to get the information of cell cycle.All values were expressed as mean±standard deviation ((X|—)+S). Results were analyzed using the Chi-square test (X² test). When p value less than 0.05 it was considered statistically significant. Use the SPSS11.0 software to analysis.Results1. For the ectopic endometrial cells corresponding to the proliferation phase of menstrual cycle, there were more cells of CO₂ group from G₀/G₁ phase entering into S and G₂/M phase than that of control group after 48h. For the secretory phase of ectopic endometrial cells, there were less cells of CO₂ group from G₀/G₁ phase entering into S and G₂/M phase than that of control group after 48h. But there were no significant differences between the groups (p>0.05).2. For the eutopic endometrial cells from EMs patients and leiomyoma of uterus patients, the cell cycles were almost the same between the treatment groups and the control groups after 24h and 48h (p>0.05).3. For EMs eutopic endometrial cells, cells at G₀/G₁ phase were fewer than that of EMs-free eutopic endometrial cells. Cells at S phase and G₂/M phase were more than that of EMs-free eutopic endometrial cells. These differences were observed at 24h, 48h after primary culture. But there were no significant differences between them (p>0.05).4. The pH value of media after the pneumoperitoneum was down to 6.2. It returned to 7.0 as before after 2 hours.5. The success rate for primary culture of ectopic endometrial cells was 63.0% (29/43), and the success rate for primary culture of eutopic endometrial cells from EMs-free women and EMs patients was 96.9% (31/32).There was a significant difference between the two rates (p<0.05).Conclusions1. The results indicated that there might be no significant effects of CO₂ pneumoperitoneum on the ectopic endometrial cells proliferation in vitro in the short term. But it was suggested that there was an increase in proliferation of ectopic endometrial cells corresponding to proliferation phase and a decrease in the prolifertaion of ectopic endometrial cells corresponding to secretory phase in vitro after CO₂ pneumoperitoneum in short term.2. It shows that there was no significant impact of CO₂ pneumoperitoneum on the eutopic endometrial cells proliferation in vitro in short term. But Proliferative activity of EMs eutopic endometrial cells was higher than that of EMs-free eutopic endometrial cells.3. It was suggested the CO₂ pneumoperitoneum could decrease the pH value of media, which was the important environment of cells in vitro.4. The primary culture of ectopic endometrial cells was more difficult than the primary culture of eutopic endometrial cells.